The Mechanism Of ZNF8-miR-552-5p Axis Modulates ACSL4-mediated Ferroptosis In Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in human beings.The onset of HCC is insidious,and many patients have lost the opportunity of surgical resection at the time of diagnosis.Although drugs such as targeted therapy and immunotherapy have brought great progress to the treatment of HCC in recent years,the overall survival of HCC patients is still a major challenge,and new treatment strategies are urgently needed.Ferroptosis is a newly discovered form of regulatory cell death characterized by the accumulation of iron ions and lipid peroxides in the cell.At the level of the fine structure of the cell,the mitochondrial membrane is wrinkled and accompanied by reduction or loss of mitochondrial crista.Studies have shown that ferroptosis is closely related to the occurrence and development of HCC.MicroRNAs(miRNAs)are a class of evolutionarily conserved non-coding RNAs that act as post-transcriptional regulators of gene expression and participate in a variety of biological processes such as cell proliferation and apoptosis.miRNAs play an important role in the occurrence and development of HCC and are considered to be a new therapeutic target for HCC.miR-552-5p is associated with a variety of malignant tumors such as breast cancer,pancreatic cancer,and gastric cancer.Studies have confirmed that miR-552-5p promotes the occurrence and development of HCC.High expression of miR-552-5p mediates sorafenib resistance in HCC by inhibiting cell apoptosis.However,whether miR-552-5p is involved in the regulation of ferroptosis in HCC is still lack of relevant studies.The aim of this study is to investigate the role and mechanism of miR-552-5p in ferroptosis,and to provide a new strategy for the treatment of HCC.Methods:The research methods adopted in this study include CCK-8,Colony formation assays,GSH assays,FerroOrange staining experiment,C11-BODIPY assay,Transmission electron microscopy(TEM)analysis,Tumor xenograft assays,Bioinformatics analysis,Western blot,Dual-luciferase reporter assay and PCR.Results:1.miR-552-5p was highly expressed in HCC.Under the condition of ferroptosis induced by RSL3,inhibition of miR-552-5p expression significantly decreased the viability of Huh-7 and Hep3B cells,decreased the level of GSH,increased the level of Fe2+,and increased the level of lipid peroxidation.Transmission electron microscopy showed that the intracellular mitochondria became smaller and the mitochondrial membrane density increased.The results were opposite after overexpression of miR-552-5p.Inhibition of miR-552-5p enhanced the sensitivity of HCC cells to ferroptosis in vivo.2.Dual luciferase reporter assay confirmed the interaction between miR-552-5p and ACSL4 mRNA 3’-UTR.The results of qPCR and Western Blot confirmed that miR-552-5p negatively regulated the expression of ACSL4.GSH assay,FerroOrange staining assay and C11-BODIPY assay confirmed that miR-552-5p reduced the sensitivity of HCC cells to ferroptosis by targeting ACSL4.3.Dual luciferase reporter assay verified the binding of transcription factor ZNF8 to miR-552-5p,and ZNF8 overexpression reduced the level of miR-552-5p in cells.CCK-8 assay,GSH assay,FerroOrange staining and C11-BODIPY assay confirmed that ZNF8 enhanced the sensitivity of HCC cells to ferroptosis by inhibiting the transcription of miR-552-5p.4.TCGA database analysis confirmed the increased expression of ACSL4 and ZNF8 in HCC,and the survival curve of HCC patients related to ACSL4 and ZNF8 expression levels was obtained through GEPIA database.The results showed that ZNF8 and ACSL4 were up-regulated in HCC tissues compared with adjacent tissues.High expression of ZNF8 as a HCC risk factor promotes HCC recurrence and reduces the overall survival rate of HCC patients,and high expression of ACSL4 as a HCC risk factor reduces the overall survival rate of HCC patients,but there is no obvious evidence that high expression of ACSL4 is significantly correlated with HCC recurrence.Conclusion:1.miR-552-5p is involved in inhibiting ferroptosis in HCC.2.miR-552-5p targeted the 3’-UTR of ACSL4 mRNA to negative regulate the expression of ACSL4.3.ZNF8 inhibits miR-552 transcription by binding to the miR-552 promoter region.4.ZNF8 and ACSL4 have reference significance for the prognosis of HCC patients.